Transcription factors represent a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes. One family of transcription factors, E2F transcription factors, regulates the expression of an extensive panel of genes that control DNA synthesis and cellular proliferation in a cell cycle-dependent manner reviewed in (Dyson, Genes Dev., 1998, 12, 2245-2262; Lavia and Jansen-Durr, BioEssays, 1999, 21, 221-230; Yamasaki, Results Probl. Cell. Differ., 1998, 22, 199-227).
The "E2F" designation given to these proteins refers to a multigene family of transcription factors that heterodimerize with members of the DP gene family (Helin and Harlow, J. Virol., 1994, 68, 5027-5035; Magae et al., J. Cell. Sci., 1996, 109, 1717-1726). E2F/DP heterodimers can mediate both transcriptional activation and repression and, to date, six members of the E2F family and two members of the DP family have been isolated.
The E2F family has been further subdivided into three subfamilies based on their interaction with the product of the retinoblastoma tumor suppressor gene (RB), a transcriptional repressor whose deregulation has been shown to result in oncogenesis. Consequently, the deregulation of the expression or function of E2F proteins alone or in conjunction with DP proteins has been implicated in the development of certain pathologic conditions, including cancer (Sladek, Cell. Prolif., 1997, 30, 97-105; Yamasaki, Biochim. Biophys. Acta, 1999, 1423, M9-15).
The first subfamily of E2Fs consists of E2F transcription factor 1, 2 and 3 (E2F-1, E2F-2 and E2F-3) which bind with high affinity to the retinoblastoma protein, pRB (Arroyo and Raychaudhuri, Nucleic Acids Res., 1992, 20, 5947-5954; Nevins, Science, 1992, 258, 424-429). The second subfamily comprises E2F transcription factors 4 and 5 (E2F-4 and E2F-5) and these bind to the pRB homologues, p107 and p130 while the third subfamily contains one member, E2F transcription factor 6 (E2F-6) that does not bind to any of the pRB family members.
While relatively little is known about the specific properties of the individual members of the E2F family, the best characterized are those of the first subfamily (E2F-1, E2F-2 and E2F-3). Because E2F binding sites are found in the promoters of many genes, these transcription factors mediate a multitude of cellular processes including apoptosis, cell cycle control, DNA replication, and proliferation (Bernards, Biochim. Biophys. Acta, 1997, 1333, M33-40; Helin, Curr. Opin. Genet. Dev., 1998, 8, 28-35; Nevins, Cell. Growth Differ., 1998, 9, 585-593).
It has been shown that when expressed alone in rat fibroblasts any of the three members of this subfamily can induce S-phase entry in serum starved cells (Lukas et al., Mol. Cell. Biol., 1996, 16, 1047-1057). In U343 astrocytoma cells, Dirks et al. have demonstrated that deregulated expression of any of the E2F transcription factors overcame cell cycle arrest and affects the expression of a distinct array of cell cycle regulatory proteins. However, it was further demonstrated that only the expression of E2F-1 or E2F-2 resulted in potent cell death (Dirks et al., Oncogene, 1998, 17, 867-876).
E2F transcription factors have been shown to regulate keratinocyte proliferation, implying a role in skin tumor development (Jones et al., J. Invest. Dermatol., 1997, 109, 187-193). In fact, it has been demonstrated that expression of five of the six members of the E2F family of transcription factors is upregulated in mouse skin tumors (Balasubramanian et al., Int. J. Oncol., 1999, 15, 387-390).
E2F transcription factor 1 (also known as E2F-1 and RBBP3 for retinoblastoma binding protein 3) was originally identified as a factor that interacts with the adenovirus early region 2(E2) promoter (Helin et al., Cell, 1992, 70, 337-350; Kaelin et al., Cell, 1992, 70, 351-364). Disclosed in U.S. Pat. No. 5,759,803 are the nucleic acid and polypeptide sequence of human E2F transcription factor 1 as well as fusion proteins and peptide fragments. Also disclosed are expression vectors encoding E2F transcription factor 1 and host cells used to express said vectors (Kaelin et al., 1998).
The role of E2F-1 in apoptosis, or programmed cell death, has been broadly investigated particularly through the use of transgenic mouse technology. Mice lacking the E2F-1 transcription factor are found to be viable and fertile yet develop a broad and unusual spectrum of tumors (Yamasaki et al., Cell, 1996, 85, 537-548) as well as exhibiting a defect in T lymphocyte development which promotes apoptosis and suppresses proliferation (Field et al., Cell, 1996, 85, 549-561). It has also been shown that the tumorigenesis induced by the loss of E2F-1 is dependent on the pRB status of the mice. In Rb1 (+/-) mice, the lifespan is lengthened and tumorigenesis reduced with the loss of the E2F-1 transcription factor, further underscoring the importance of the regulated control of E2F-1 expression (Yamasaki et al., Nat. Genet., 1998, 18, 360-364).
Despite similarities among E2F members, distinct functional roles have been identified in certain cell types. In studies of hematopoiesis, overexpression of E2F-1 in myeloid progenitor cells was shown to override survival signals in the cell provided by growth factors and lead to apoptosis. In contrast, overexpression of E2F-3 did not lead to apoptosis but to normal granulocyte differentiation (Strom et al., Cell Growth Differ., 1998, 9, 59-69).
E2F-1 expression has been shown to be upregulated in response to DNA damage in much the same manner as p53, a known tumor suppressor (Blattner et al., Mol. Cell. Biol., 1999, 19, 3704-3713). E2F-1 has also been shown to physically interact with p53 and target the protein for rapid degradation. The pharmacological modulation of E2F transcription factor 1 expression and/or function may therefore be an appropriate point of therapeutic intervention in pathological conditions involving the loss of tumor suppressor genes.
Gene amplification of the E2F-1 locus has also been identified in several established human leukemia cell lines further supporting a role for E2F-1 in tumorigenesis (Saito et al., Genomics, 1995, 25, 130-138).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of E2F transcription factor 1 and investigative strategies aimed at modulating E2F transcription factor 1 function have involved the use of antibodies, molecules that block upstream entities, antisense expression vectors, antisense techniques, decoy oligonucleotides, chemical inhibitors and gene knock-outs in mice.
Generally disclosed in the PCT publication WO 95/24223 are antisense oligonucleotides specific for E2F-1 for use in the treatment of neoplastic disease. No specific antisense oligonucleotide sequences are given, however (Calabretta, 1995).
The use of decoy oligonucleotides that block the binding of the E2F transcription factor with the target regions along gene promoters are also reported in the literature. These decoy oligonucleotides have been investigated as therapies for vascular proliferative disorders such as atherosclerosis, postangioplasty restenosis and vein graft disease (Mann, Antisense Nucleic Acid Drug Dev., 1998, 8, 171-176; Tomita et al., Am. J. Physiol., 1998, 275, F278-284). There remains, however, a long felt need for additional agents capable of effectively inhibiting E2F transcription factor 1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of E2F transcription factor 1 expression.
The present invention provides compositions and methods for modulating E2F transcription factor 1 expression.